Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure and assembly of macromolecular complexes. This year, we completed and published studies linking protein folding and ultrafast protein solvation. We continued and expanded our femtosecond upconversion studies of Trp in proteins and peptides to quantify early quasistatic self-quenching processes that confound studies of solvation dynamics. We previously published evidence that extremely rapid (10-100ps) decays are important in several proteins (crystallins, thioredoxin, etc.), as they detect previously silent conformers engaged in ultrafast charge transfer. Our earlier study of protein *solvation* on the 330fs-200ps time scale, using proteins such as Monellin, found QSSQ that others attributed to a class of unique water molecules that desorb from protein in 20ps. Local quenching is the dominant mechanism in all but a few cases we have studied. The QSSQ was found even in simple dipeptides ,suggesting a general process underlies all protein QSSQ. We explained the slow relaxation (50ps) of water in the protein GB1 as a function of pH and temperature, helping to understand why this one small protein lacks the QSSQ that usually masks relaxation. At the same time, we have determined the relaxation is not coupled to local solvent access of Trp (measured during pH titration with soluble quenchers and also seen in ns lifetimes). This also showed that the charge environment changes accompanying titration did not change the local mobility of water. We also completed studies using Trp analogs that , almost immune to QSSQ, respond to relaxing water matrices without masking by local electron transfer kinetics. The titration study was published; the overall subject of upconversion (sub-ps) studies of Trp was separately published by us in a book chapter, and the analog experiments we published showed concurrent relaxation and QSSQ may occur. We contined collaborative studies with LCE into the status of a primary fuel of heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and in collaboration with Light Microscopy Core and LCE, we are continually refining 'Decay-Associated Images' software to more rapidly extract profiles of NADH binding within isolated cardiac myocytes and/or tumor cells. This year we initiated NADH studies in basal cell carcinoma or melanoma models, and we again updated DAI software for large images. We continue to develop coupled lifetime and translational diffusion capabilities in time-resolved FCS for this and other projects.